Filtration of airborne eDNA outperforms leaf swabs for terrestrial vertebrate detection in a temperate mixed forest with longer filtration times performing the best

Advances in collection of environmental DNA (eDNA) through active air filtration and sampling from vegetation (leaf swabs) have demonstrated the promise of using airborne eDNA for terrestrial vertebrate surveys. However, methods are at an early stage, and we lack insights into the effect of air filtration time and the complementarity between the two sampling methods for characterising vertebrate communities. Here we filtered airborne eDNA for 24, 48, and 96 h in a temperate forest in Denmark and used DNA metabarcoding of a 12S bird and a 16S mammal marker to assess how the duration of active airborne eDNA filtration affects vertebrate detections. Further, we assessed the complementarity between 24‐h active airborne eDNA filtration and leaf swabs. We found a strong positive correlation between air filtration time and taxa richness. On average, we detected 24 wild vertebrate taxa per sample with 96‐h sampling, while 24‐h sampling yielded an average of 15 wild taxa per sample. Increased sampling time also led to characterisation of more homogeneous communities across samples. However, 24‐h sampling yielded the highest total number of wild taxa (66 taxa), with 48‐ and 96‐h sampling capturing 60 and 55 taxa, respectively. As we collected forty 24‐h samples, twenty 48‐h samples, and ten 96‐h samples, this may reflect the number of analysed samples. Compared with filtered air samples, leaf swabs yielded fewer vertebrate taxa per sample (average 1.5). In summary, air filtration outperformed leaf swabbing and longer air filtration time yielded a more diverse and consistent vertebrate community in a temperate forest. This provides insights for the use of active filtration of airborne eDNA and leaf swabbing for biodiversity surveys, ecological studies, conservation, nature management programmes, and beyond.